GLUT4 is the isoform principally responsible for insulin-mediated glucose uptake in mammalian tissues. Glucose homeostasis is sensitive to changes in GLUT4 levels. Modulation of GLUT4 levels is therefore an attractive molecular target for therapeutic intervention in insulin-resistant states, including diabetes mellitus. A straightforward approach to enhance GLUT4 expression is to increase the transcription rate of the gene. GLUT4 gene expression is transcriptionally regulated in physiologic states such as insulin-deficiency and exercise, and it is likely that a pharmacological intervention can be developed to enhance Glut4 gene transcription. To reach this goal, we must first understand the molecular basis for transcriptional regulation of the GLUT4 gene. Using transgenic mice, we have shown that cis-elements regulating the human Glut4 promoter are located within 895 bp immediately 5' of the transcription initiation site. This region contains two major regulatory domains, referred to as Domain I and the MEF2 domain. These elements function cooperatively to support regulated expression of GLUT4. The MEF2 domain binds isoforms of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors, while Domain I binds GEF (GLUT4 Enhancer Factor), a transcriptional activator identified and cloned in our laboratory. MEF2 isoforms and GEF form a protein complex in vivo; however, the function of this complex in regulating gene transcription is not known. A conserved Liver Receptor X (LXRE) domain is located immediately adjacent to the MEF2 site. The functional role of this element has not been determined. We hypothesize that both the tissue-specific and the hormonal and/or metabolic regulation of the GLUT4 gene are carried out through these regulatory domains and their cognate binding proteins. The primary goal of this proposal is to understand the molecular mechanisms of the tissue-specific and hormonal/metabolic regulation of GLUT4 gene transcription. To achieve these goals, we propose the following specific aims: 1) To determine the functional role of GEF in GLUT4 transcriptional activation; 2) To understand the role of HDAC function in regulation of GLUT4 expression 3) ) To determine the contribution of the GLUT4 LXRE in mediating metabolic signals to the GLUT4 promoter. Completion of these aims will inform us what signals changes in GLUT4 expression in a variety of altered metabolic conditions of insulin- deficiency or insulin resistance. These conditions each result in down-regulation of GLUT4 expression, but it is unclear if this is by a single mechanism. Completion of the aims of this proposal will help us to understand more about the GLUT4 promoter and more about the intracellular signaling during insulin- deficiency and insulin-resistance. PUBLIC HEALTH RELEVANCE: Diseases characterized by insulin resistance such as diabetes mellitus and metabolic syndrome are increasingly prevalent around the world. Animal models and human clinical studies suggest that increasing the production of the insulin- responsive glucose transporter (GLUT4) protein is a good way to treat these diseases. The research goals of this project are very important for learning how we can increase the production of the GLUT4 protein. This work will facilitate drug discovery to achieve this desired goal.